Chemiluminescence immunoassay is a non-radioactive immunoassay produced using a labeling illuminant as a tracer signal, which has the advantages of high sensitivity, broad linear range, rapid analysis speed, simple operation, and ready for automation.
Currently, chemiluminescence immunoreagents commonly used in clinical practice are:
1. Reaction systems using alkaline phosphatase and horseradish peroxidase with luminol. The main disadvantage thereof is that the activities of alkaline phosphatase and horseradish peroxidase are greatly affected by temperature variation, and the experimental results are greatly affected by environmental variation.
2. Luminescence reactions using alkaline solution of acridinium ester and derivatives thereof with hydrogen peroxide. The main disadvantage is that it is a flash luminescence with short luminescence time, which can only be detected on a fully automated instrument. The mode for the calculation of the results is complex, and the experimental results are not stable.
3. Electro-chemiluminescence systems using ruthenium pyridine and tripropylamine.
Electro-chemiluminescence (ECL) reaction is a specific chemiluminescence reaction triggered by electrochemistry on the surface of an electrode. The conjugate of antigen-antibody complex and ruthenium pyridine is electrochemically excited by electrochemistry in the presence of tripropylamine, and a redox reaction occurs to emit photons, which can be collected by a photomultiplier tube. This process is performed repeatedly to produce plenty of photons, which enhance the optical signal. The labels commonly used in the electrochemiluminescence analysis are those which can bind to an antibody or an antigen molecule with different chemical structures, to produce a labeled antibody or antigen.
Ruthenium pyridine is water-soluble and highly stable, which ensures an efficient and stable electrochemiluminescence reaction and avoids the interference from background noise. Even when the molecular ratio of the binding of ruthenium pyridine to immunoglobulin exceeds 20, the solubility and the immunological activity of antibodies are not affected, the molecular weight and steric hindrance remain low, and thus even a small molecular nucleic acid can also be labeled.
The currently available electrochemiluminescence methods mainly employ tripropylamine (TPA) as a reactant. The disadvantages of tripropylamine are that the reaction is slow, the concentration is high, it is greatly affected by electrode material, the luminescence efficiency is limited, and it is not cost optimized.